Exogenous C2-ceramide activates c-fos serum response element via Rac-dependent signalling pathway.
Ceramide is an important regulatory molecule implicated in a variety of biological processes in response to stress and cytokines. To understand the signal transduction pathway of ceramide to the nucleus, in the present study, we examined whether C2-ceramide, a cell permeable ceramide, activates c-fos serum response element (SRE). Treatment of Rat-2 fibroblast cells with C2-ceramide caused the stimulation of c-fos SRE-dependent reporter gene activity in a dose- and time-dependent manner by transient transfection analysis. Next, we examined the role of Rho family GTPases in the ceramide-induced signalling to SRE activation. By reporter gene analysis following transient transfections with various plasmids expressing a dominant negative mutant form of Cdc42, Rac1 or RhoA, C2-ceramide-induced SRE activation was shown to be selectively repressed by pEXV-RacN17 encoding a dominant negative mutant of Rac1, suggesting that Rac activity is essential for the signalling cascade of ceramide to the nucleus. In a further study to analyse the downstream mediator of Rac in the ceramide-signalling pathway, we observed that either pretreatment with mepacrine, a potent and specific inhibitor of phospholipase A2, or co-transfection with antisense cytosolic phospholipase A2 (cPLA2) oligonucleotide repressed the C2-ceramide-induced SRE activation selectively, implying a critical role of cPLA2 in C2-ceramide-induced signalling to nucleus. Consistent with these results, the translocation of cPLA2 protein as well as the release of arachidonic acid, a principal product of phospholipase A2, was rapidly induced by the addition of C2-ceramide in a Rac-dependent manner. Together, our findings suggest the critical role of 'Rac and subsequent activation of phospholipase A2' in ceramide-signalling to nucleus.
Kim
B C
BC
Laboratory of Molecular & Cellular Genetics, Institute of Environment and Life Science, Hallym University, Chun-Cheon, Kangwon-do, South Korea #200-702.
Kim
J H
JH
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005347:Fibroblasts / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D051381:Rats; D026362:Serum Response Factor; D015398:Signal Transduction; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D020744:rac GTP-Binding Proteins
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C064769:N-acetylsphingosine; D009687:Nuclear Proteins; D026362:Serum Response Factor; D014157:Transcription Factors; D016718:Arachidonic Acid; D010741:Phospholipases A; D054467:Phospholipases A2; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins; D013110:Sphingosine